Human B cells

B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype.[1] They function in the humoral immunity component of the adaptive immune system by secreting antibodies.[1] Additionally, B cells are also classified as professional antigen-presenting cells (APCs) and secrete cytokines (wikipedia)
Throughout life, B cells are generated from lymphoid precursors. B-cell development takes place in the bone marrow in a tightly regulated process, with stepwise recombination of V, (D) and J gene segments coding for the variable (V) region of the immunoglobulin (Ig) heavy and light chains.
After the expression of a functional B cell receptor, naïve B cells leave the bone marrow and circulate though the body via the blood steam. Upon activation B cells can either develop into plasma cells or migrate into secondary lymphoid organs like tonsil, spleen or Payer’s patches for further differentiation. Mainly naïve B cells also migrate through non-lymphoid tissue.
Below we describe how B cells from blood, bone marrow and tissue can be isolated, taken into culture and analysed by flow cytometry.

B cells circulate through the body via the peripheral blood. Among all peripheral blood lymphocytes, B cells account for 2-10% of all lymphocytes. All mature B cells in blood express the Pan B cell marker CD19, CD20 and CD22.
Peripheral blood B cells can be classified into transitional/immature, naïve and memory B cells and plasma cells. Additionally, different subsets of memory B cells and plasma cells can be identified based on their expression of their Ig isotypes (IgM, IgD, IgG, IgA).

5.1.1.1 Naïve B Cells

If a B cell succeeds in producing a functional, non-autoreactive BCR, it differentiates into a mature, naive B cell. These cells express the BCR as IgM and IgD molecules, recirculate through the body and account for ~50% of B cells in adults. Naïve B cells are negative for CD27.

5.1.1.2 Memory B cells

Memory B cells are generated in germinal center (GC) reactions in the course of T cell-dependent immune responses and are distinguished from naive B cells by an increased lifespan, faster and stronger response to stimulation and expression of somatically mutated and affinity matured immunoglobulin (Ig) genes. Approximately 40% of human B cells in adults are memory B cells, and several subsets were identified. Besides IgG+ and IgA+ memory B cells, ∼50% of peripheral blood memory B cells express IgM with or without IgD. Further smaller subpopulations have additionally been described. These various subsets share typical memory B cell features, but likely also fulfill distinct functions. 
In humans, the tumor necrosis factor receptor superfamily member CD27 became an important marker to distinguish naive from mutated IgM+ B cells (note that GC B cells and post-GC PCs are also CD27+).2, 36

IgG memory B cells

Approximately 15–20% of peripheral blood (PB) B cells in adults are IgG+ B cells, expressing mainly IgG1, IgG2 or IgG3, whereas IgG4 is very rare.37 Their proportions among memory B cells vary considerably among individuals, likely because of different infection histories, as switching to specific IgG subclasses is influenced by the type of antigen and various immunoregulatory factors. 
Approximately 20–25% of IgG memory B cells lack CD27 expression.37 The increase of this subpopulation in the elderly has led to the idea that these cells are aged or exhausted memory B cells.38 IgG+CD27− B cells are considerably less mutated and are more frequently IgG3+ and less frequently IgG2+.27, 39 However, both types of memory B cells often derive from common GC B-cell clones.27, 39 The specific characteristics of the two IgG+ memory B-cell subsets indicate their common generation, but different kinetics or dependency on different microenvironmental stimulations.
In mice and humans, IgG memory B cells have the propensity to differentiate into PCs upon reactivation, and mostly not to reenter GC reactions.31, 32, 33 

IgA memory B cells

IgA memory B cells account for ~10% of B cells in the PB and mostly express CD27. They are preferentially generated in immune responses in the intestine and other mucosa-associated lymphatic tissues, and predominantly localize 
IgE-expressing memory B cells are hardly detectable in the PB of healthy humans.

IgM-only and IgM+IgD+ memory B cell

Two distinct subpopulations of human IgM-expressing PB B cells with somatically mutated IgV genes exist, IgM-only B cells (IgM+IgDlow/−) and IgM+IgD+ B cells, both expressing CD27 and representing ~5% and 15% of B cells, respectively, in PB and secondary lymphoid organs, hence representing a major fraction of the B-cell pool.2, 17, 49

taken from Leukemia. 2016 Dec;30(12):2283-2292. doi: 10.1038/leu.2016.226. Epub 2016 Aug 8.
Human memory B cells. Seifert M1, Küppers R1. https://www.ncbi.nlm.nih.gov/pubmed/27499139 

5.1.1.3 Plasma cell

Human plasma cells are a heterogenous cell population comprising several subsets from short-lived and proliferative plasmablasts in lymphoid tissues, transitional plasma cells in peripheral blood to long-lived and non-dividing plasma cells in bone marrow. Plasma cells of all differentiation stages are identified by the expression of high levels of the CD38 antigen. Other surface markers, however, are differentially regulated dependent on the stage of differentiation and the spatial localization. Plasma cells in the blood circulation lack the expression of the typical B cell marker CD22 and were found to express lower levels of CD19 than mature B cells. They are further characterized as being CD27++CD31+CD44+CD45+CD56–CD62L+CD86+ and HLA-DR+. A subset of CD38+ blood plasma cells further expresses the CD138 antigen, whereas all CD38+ plasma cells are also CD138+ in bone marrow. (MB webpage)

5.1.1.4 Activated B cells

B Cell Expansion
B cell frequency in human peripheral blood is often too low to obtain sufficient cell numbers for research purposes. Therefore, B cell expansion is an important step in human B cell research. Usually B Cell expansion is achieved by stimulation of CD40. CD40L-CD40 interaction is important in T cell-APC (antigen-presenting cell) interaction and is e.g. involved in B cell differentiation and proliferation, isotype class-switching, and protection of B cells from apoptosis. 
The B cell Expansion Kit offers the possibility to expand B cells up to 200 times in 14 days. The Kit contains the CD40-Ligand and a cross-linking antibody for multimerization to increase the biological activity of CD40-Ligand, as well as premium grade Human IL-4 and StemMACS HSC Expansion Medium XF, human. The B cell expansion is achieved by culturing and restimulation at day 7 and 10 of culture.

5.1.5.1 Flow cytometric analysis of cell surface marker

B Cells can be analysed by flow cytometry by different extracellular as well as intracellular markers. 
Flow cytometric analysis of surface markers
The MC CD19 B Cell Cocktail is designed to enable easy and rapid evaluation of MACS Separations using either CD19 MicroBeads, CD20 MicroBeads or the B Cell Isolation Kit II. However, it is also suitable to analyse peripheral blood B cells without separating them before.
MC CD19 B Cell Cocktail consists of CD19 and CD20 antibodies for reliable identification of B cells. In addition, a VioBlue-conjugated CD45 antibody is included as a trigger to restrict analysis to leukocytes. VioBlue requires the use of a violet laser.
Either this Pre-mixed antibodies can be used or any other combination of markers and dyes.
For the analysis of the peripheral blood B cell subsets, the following marker combination is appropriate for a clear identification of the respective subset. 

Overview on all available antibodies:
http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/antibodies-and-dyes.aspx


5.1.5.2 Cytokine detection

 Like T cells, B cells regulate the immune response by the secretion of various cytokines. Cytokine secretion can be measures in different ways.
MACS Cytokine Secretion Assays are designed for highly sensitive detection of viable cytokine-secreting cells. This technology allows the detection of secreted cytokines at a single-cell level and simultaneous phenotyping of cytokine-secreting cells.
link to Detail page Technology:  CSA 
For the optional enrichment of viable cytokine-secreting cells Miltenyi Biotec offers the Cytokine Secretion Assay – Cell Enrichment and Detection Kits. 
However,  B cells are not homogenous with respect to cytokine production.  B cells primed by Th1 cells and antigen (Be-1 cells) make cytokines associated with type 1 immune responses, such as IFNγ and IL-12, while B cells primed by Th2 cells and antigen (Be-2 cells) make IL-2, IL-13 and IL-4. So far, regulatory B cells are identified by their ability to secrete IL-10 or TGFβ-1.
For an overview on all offered Cytokine Secretion Assays go to: http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/kits-and-assays.aspx